High-density lipoprotein receptor SCARB1 is required for carotenoid coloration in birds

Abstract

Yellow, orange, and red coloration is a fundamental aspect of avian diversity and serves as an important signal in mate choice and aggressive interactions. This coloration is often produced through the deposition of diet-derived carotenoid pigments, yet the mechanisms of carotenoid uptake and transport are not well-understood. The white recessive breed of the common canary (Serinus canaria), which carries an autosomal recessive mutation that renders its plumage pure white, provides a unique opportunity to investigate mechanisms of carotenoid coloration. We carried out detailed genomic and biochemical analyses comparing the white recessive with yellow and red breeds of canaries. Biochemical analysis revealed that carotenoids are absent or at very low concentrations in feathers and several tissues of white recessive canaries, consistent with a genetic defect in carotenoid uptake. Using a combination of genetic mapping approaches, we show that the white recessive allele is due to a splice donor site mutation in the scavenger receptor B1 (SCARB1; also known as SR-B1) gene. This mutation results in abnormal splicing, with the most abundant transcript lacking exon 4. Through functional assays, we further demonstrate that wild-type SCARB1 promotes cellular uptake of carotenoids but that this function is lost in the predominant mutant isoform in white recessive canaries. Our results indicate that SCARB1 is an essential mediator of the expression of carotenoid-based coloration in birds, and suggest a potential link between visual displays and lipid metabolism.

Keywords: Serinus canaria; carotenoids; coloration; lipid metabolism.

Fig. 1.

White recessive canary has white plumage and very low carotenoid levels in its tissues. (A and B) Representative images of the (A) lipochrome domestic canary and (B) white recessive canary. (C) Carotenoid concentrations of the liver, retina, skin, and feathers of typical yellow (yellow points) and white recessive (WR; open points) canaries. The lines represent the means for each breed and tissue. Carotenoid concentration was calculated relative to protein content in liver, retina, and skin samples, or a dry mass of feathers. All carotenoid types within a given sample were summed to give the total concentration.

Fig. 2.

Mapping of the white recessive mutation. (A) Selective sweep mapping. F_ST between white recessive and nonwhite breeds/populations across the autosomal scaffolds. Each dot represents F_ST in 20-kb windows iterated every 10 kb. The different scaffolds are presented along the x axis in the same order as they appear in the canary reference genome assembly. (B) F_ST zoom-in and IBD mapping. (Top) F_ST in 20-kb windows iterated every 10 kb across the outlier region (delineated by vertical lines). (Bottom) The protein-coding genes found within this region are indicated by green boxes, and black lines point to the position of the genotyped SNPs in the IBD analysis. For the IBD analysis, 38 SNPs were genotyped for 24 white recessive canaries and 61 individuals belonging to 8 breeds with yellow or red coloration. Alleles more common in white recessive canaries are represented by white boxes, alternative alleles are in yellow, and heterozygotes are in orange. The red-outlined boxes indicate a segment of high homozygosity in white recessive canaries and black boxes indicate missing data.

Fig. 3.

Splicing variants and expression levels of SCARB1 in white recessive and wild-type individuals. (A) SCARB1 contains alternative splice donor sites that potentially yield different transcript isoforms. To investigate these alternatives, we designed primers (fragment) to amplify across this region, yielding different amplicon sizes from each isoform (B). To quantify the abundance of each alternative isoform (C), we designed qPCR primers spanning each potential splice junction (dashed lines). (B) Capillary electrophoresis fragment analyses of amplicons across the exon 4 splice junctions, generated from skin cDNA of white recessive or yellow canaries, indicate that three alternative SCARB1 transcript isoforms are present in the skin of the white recessive canary. This multitemplate amplification is biased toward short amplicons, and therefore the intensity of the peaks does not necessarily reflect transcript abundance. a.u., arbitrary units. (C) The mean ± SD of relative expression of SCARB1 transcript isoforms in the duodenum, liver, eye, and skin of yellow (n = 4; yellow bars) and white recessive (n = 4; white bars) canaries. For each sample, expression was measured by qPCR and calculated relative to a transcript region 3′ of the splice sites (exon 7/8) and then normalized to the sum of the four isoforms. The asterisk indicates a significant difference between yellow and white recessive (P ≤ 0.045).

Fig. 4.

Analysis of the carotenoid transport function of SCARB1 splice isoforms. Here are presented the total carotenoid concentration (µg carotenoid per g of protein) of avian fibroblast cells expressing wild-type SCARB1 (yellow points), the exon 4-deficient isoform (isoform 4) of SCARB1 (open points), or a fluorescent protein-only control (black points). The cells were supplemented with carotenoid-containing whole-chicken serum to a final concentration of 0.81 μg⋅mL⁻¹ of carotenoid in media (experiment 1) or 1 μg⋅mL⁻¹ of pure zeaxanthin solubilized with Tween 40 (experiment 2). The lines represent the mean for each condition within each experiment. Significant differences (P ≤ 0.006) between conditions are denoted with an asterisk.